Vaccination with tumor-derived GRP94/gp96, the endoplasmic reticulum Hsp90 molecular chaperone, can elicit suppression of tumor growth and metastasis. The substantial therapeutic efficacy of GRP94/gp96 in prophylactic and therapeutic vaccination of mice has prompted its clinical evaluation as an immunotherapeutic for treatment of cancer in humans. Currently, GRP94/gp96 is undergoing Phase III trials for kidney cancer and melanoma. Although GRP94/gp96 has generated substantial interest as a novel immunotherapeutic, its mechanism of action remains unknown. The broad, long-term objective of the proposed research is to identify the mechanism of GRP94/gp96-mediated anti-tumor immune responses. This objective will be accomplished in the following specific aims: 1. Define the immunological basis for a gp96-secreting, cell-based tumor vaccine strategy; 2. Determine the role of gp96 and gp96 NTD in the regulation of innate immune responses. 3. Examine the role of gp96 and gp96 NTD in early immune modulation of retrovirus-induced disease. 4. Identify the effector cells and effector cell receptor(s) functioning in gp96/gp96NTD recognition and cell activation. The proposed studies will utilize a recently developed novel immunization strategy wherein animals are vaccinated with cells expressing a secretory form of GRP94/gp96. Using this experimental approach, we have demonstrated that GRP94/gp96-mediated tumor rejection is independent of the tissue of origin and thusGRP94/gp96-elicited anti-tumor immune responses can be elicited using GRP94/gp96 of non-tumor origin. Given the absence of tumor-restriction identified in these experiments, we hypothesize that GRP94/gp96functions through activation of innate immune responses. Such responses are a necessary prerequisite to robust anti-cancer adaptive immune responses. In executing the Specific Aims presented above, we will define the efficacy of the gp96-secreting, cell-based tumor vaccine strategy in multiple tumor models, using prophylactic and therapeutic immunization strategies. Detailed studies of the interactions of GRP94/gp96 with effector cells of the innate immune system will be performed. These studies will emphasize in vivo models and analyses of in vivo cellular responses. To better define the mechanism of GRP94/gp96 interaction with the innate immune system, we propose to extend analyses to the study of retrovirus-induced disease, in a neonate model, and to identification of signaling pathways accessed by GRP94/gp96.